Analysis of immune complexes in rheumatoid arthritis for Epstein-Barr virus antigens reveals cross-reactivity of viral capsid antigen and human IgG

We recently defined the immunochemical characteristics of immune complexes (IC) isolated from synovial fluid (SF) of patients with rheumatoid arthritis with the use of Western blot analysis. In the present study, we probe for exogenous antigens in the IC by examining the specificity of antisera raised against the IC. Anti-IC antisera demonstrated strong reactivity against the viral capsid antigen (VCA) of Epstein Barr virus (EBV), which was not explained by preimmune reactivity, polyclonal B cell activation, or Fc-mediated binding in the immunofluorescence or ELISA systems used to measure antibody titers. However, comparable anti-VCA reactivity was detected in antisera raised against non-rheumatoid SF. This phenomenon was not due to antigen since monoclonal anti-VCA antibody probing the IC by Western blot detected only IgG, nor to idiotype/anti-idiotype interaction since normal IgG absorbed out the anti-VCA reactivity. A monoclonal anti-VCA antibody competitively inhibited the binding of anti-IgG to IgG, and Fc fragment of IgG competitively inhibited the monoclonal antibody binding to VCA. No relationship between IgG anti-VCA antibody and IgG rheumatoid factor could be demonstrated. These data demonstrate an unexpected cross-reactivity of Fc fragment of IgG and VCA of EBV through the analysis of SF IC.     

The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation,but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.     

Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein

Mouse monoclonal antibody MON-100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON-100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON-100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON-100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON-100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of Mr 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti-7B2 monoclonal antibody MON-100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments.     

Specificity of the localization of transforming growth factor-alpha immunoreactivity in colon mucosa.

Transforming growth factor-alpha (TGF-alpha) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-alpha immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-alpha knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-alpha) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-alpha) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-alpha knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-alpha molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-alpha should be used for TGF-alpha immunohistochemistry in the colon.     

Monoclonal antibody MS13 identifies a plasmatocyte membrane protein and inhibits encapsulation and spreading reactions of Manduca sexta hemocytes

Lepidopterans generally can successfully defend themselves against a variety of parasites or parasitoids. One mechanism they use is to encapsulate the invader in many layers of hemocytes. For encapsulation to occur, the hemocytes must attach to the foreign material, spread, and adhere to each other. The molecules that mediate these processes are not known. One method to identify proteins potentially necessary for adhesion, spreading, and, thus, encapsulation is to use monoclonal antibodies that interfere with these functions. In this paper, we report that a monoclonal antibody against Manduca sexta plasmatocytes effectively inhibited encapsulation of synthetic beads in vitro and in vivo. Furthermore, it inhibited plasmatocyte spreading in vitro. Other anti-hemocyte antibodies did not have these effects. The plasmatocyte-specific monoclonal antibody, mAb MS13, recognized a protein of approximately 90,000 daltons as indicated by Western blot analysis of hemocyte lysate proteins. The epitope recognized by mAb MS13 was present on the exterior surface of plasmatocytes. Using indirect immunohistochemistry with hemocyte-specific antibodies, we also determined that during encapsulation plasmatocytes were the first cells bound to latex beads and later layers consisted of both plasmatocytes and granular cells. Arch.     

Effects of Anti-Glutamate-Binding Protein Antibodies on Synaptic Membrane Ion Flux, Glutamate Transport and Release, and l-Glutamate Binding Activities

Antibodies (Abs) raised against the L-glutamate-binding protein (GBP) purified from bovine brain were used to define the possible physiologic activity of GBP in synaptic membranes. Three processes were examined for their sensitivity to the Abs: the excitatory amino acid stimulation of thiocyanate (SCN-) flux, the transport of L-glutamic acid across the synaptic membrane, and the depolarization-induced release of L-glutamate. Only the amino acid-induced changes in ion flux were inhibited by the anti-GBP Abs. The change in membrane potential produced by exposure of synaptic membranes to excitatory amino acids was measured as the increase in the uptake of the lipophilic anion SCN-. The L-glutamate-induced SCN- influx was 40 times more sensitive to inhibition by the anti-GBP Abs than the stimulation of ion flux by kainate, and 60 times more sensitive than that produced by quisqualate. The anti-GBP Abs did not inhibit the activation of ion flux produced by N-methyl-D-aspartate. The inhibition of glutamate-stimulated ion fluxes by the Abs was complete, whereas the inhibition of L-glutamate binding to either the rat or bovine brain GBP was not. The results obtained indicated that although the majority of the anti-GBP Abs were not directed against the glutamate recognition site of the GBP and of presumed synaptic membrane receptors, they were effective in blocking the activation of receptor-associated ion channels. Thus, the GBP may be considered a component of some excitatory amino acid receptor complexes.     

疟原虫抗原B表位NKND在鼠伤寒沙门氏菌鞭毛蛋白中的表达

疟原虫抗原B表位NKND是由本课题组首先报道的一个新的疟疾抗原表位．它是存在于多种不同疟原虫分离株中的保守序列．以减毒沙门氏菌为载体制备口服活疫苗,方法简单,使用方便,其鞭毛蛋白有较强的免疫原性,有利于激发人体的细胞和体液免疫．人工合成8肽的实验中[2]发现NKNDD可增强NKND对相应抗体的亲和力．将人工合成的NKNDD寡核苷酸片段插入鼠伤寒沙门氏菌鞭毛蛋白表达体系中,经Western杂交鉴定,表达出具有多拷贝NKNDD表位的重组鞭毛蛋白．     

The monoclonal antibody 2G8 is carbohydrate-specific and distinguishes between different forms of vertebrate cholinesterases

The monoclonal antibody (mAb) 2G8 (subclass IgG2a) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys flesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1.8) from human serum. Binding of mAb 2G8 to the globular G2 form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC.

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Identification of human hsp-70 expression in stably transfected rodent cells by isoelectrofocus immunoblots.

The major heat-shock protein, hsp-70, is synthesized by cells from a wide variety of organisms in response to heat shock or other stresses. It is assumed that hsp-70 may have an important thermal protective function. To test this hypothesis directly, we have transfected rat fibroblast cells with appropriate expression plasmids containing a cloned human hsp-70 gene. Stable transfectants expressing the human hsp-70 gene product were identified by Western blot analysis. During the course of selecting successful transfectants, we found that when standard methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining were employed with the monoclonal antibody raised against the human hsp-70 antigen, we were unable to differentiate the human hsp-70 from the heat-inducible rat hsp-70. This was because the monoclonal antibody cross-reacts with the human and rat proteins, which have the same mobility in SDS-PAGE, and it is difficult to determine which protein is expressed. To improve the resolution of the Western blot technique, we performed additional immunoblot analysis of cellular proteins separated by slab gel isoelectrofocusing. Our study shows that the isoelectrofocusing technique, when combined with antibodies against hsp-70, gave a better resolution for the separation of exogenous human hsp-70 and the endogenous rat hsp-70 than the commonly used SDS-PAGE Western blot analysis. It provides a rapid and specific method to identify positive colonies that express the human hsp-70 gene in transfected rodent cells.     

Generation and characterization of a monoclonal antibody specific for the major thiol-activated cysteine proteinase of Porphyromonas gingivalis W83

An IgM monoclonal antibody specified for the thiol-activated proteinase of the oral pathogen Porphyromonas gingivalis W83 was generated. The antibody reacted with a single protein of approximate molecular mass 43 kDa in outer membrane preparations of P. gingivalis. Immuno-electron microscopy using the monoclonal antibody and immunogold labelling confirmed the cell surface location of the thiol-activated proteinase. The monoclonal antibody failed to detect any proteins in Western blot analysis of other closely related oral bacteria. The specificity of this monoclonal antibody to the thiol-activated proteinase of P. gingivalis should allow its use as a diagnostic tool for the rapid enumeration of P. gingivalis in clinical samples.     

Monoclonal antibody to GABA binding protein, a possible GABAB receptor.

A monoclonal antibody has been raised against a partially purified preparation for the GABAB receptor. The antibody recognized a protein of about 80 kDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove, without visible changes in electrophoresed profiles of total proteins, over 90% of the baclofen suppressive GABA binding activity (designated herein, GABAB receptor binding activity) in the solubilized synaptic membrane fraction. Moreover, the addition of GB-1 antibody directly inhibited the GABA binding activity in the crude synaptic membrane fraction. These results indicate that the monoclonal antibody obtained here recognizes the GABA binding protein, or more specifically a GABAB receptor.     

Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.     

Use of monoclonal antibodies against dibenzo-p-dioxin degrading Sphingomonas sp. strain RW1

I.S. THAKUR. 1996. A monoclonal antibody prepared against surface antigen of Sphingomonas sp. strain RW1 was used for the direct detection of RW1-like organisms in environmental samples by epifluorescence microscopy and subsequent confirmation by Western blot. Of the 76 samples collected from various sources and probed using epifluorescence, only one sample, effluent from paper and pulp processing, gave a positive result. The effluent was cultured and yielded an organism which, by Western blot analysis, was shown to contain the 28 kDa protein recognized by the monoclonal antibody.     

Invertebrate acetylcholinesterases: Insights into their evolution and non-classical functions

Acetylcholinesterase (AChE) plays a pivotal role in synaptic transmission by hydrolyzing the neurotransmitter acetylcholine. In addition to the classical function of AChE in synaptic transmission, various non-classical functions have been elucidated. Unlike vertebrates possessing a single AChE gene (ace), invertebrates (nematodes, arachnids, and insects) have multiple ace loci, encoding diverse AChEs with a range of different functions. In the field of toxicology, AChE with synaptic function has long been exploited as the target of organophosphorus and cabarmate pesticides to control invertebrate pests for the past several decades. However, many aspects of the evolution and non-classical roles of invertebrate AChEs are still unclear. Although currently available information on invertebrate AChEs is fragmented, we reviewed the recent findings on their evolutionary status, molecular/biochemical properties, and deduced non-classical (non-neuronal) functions.     

Detection of circulating and endothelial cell polymers of Z and wild type alpha 1-antitrypsin by a monoclonal antibody

Globular inclusions of abnormal alpha1-antitrypsin (AAT) in the endoplasmic reticulum of hepatocytes are a characteristic feature of AAT deficiency of the PiZZ phenotype. Monoclonal antibodies, which contain constant specificity and affinity, are often used for the identification of Z-mutation carriers. A mouse monoclonal antibody (ATZ11) raised against PiZZ hepatocytic AAT was successfully used in enzyme-linked immunosorbent assays (ELISA) and in identification of Z-related AAT globular inclusions by immunohistochemical techniques. Using electrophoresis, Western blotting, and ELISA procedures, we have shown in the present study that this monoclonal antibody specifically detects a conformation-dependent neoepitope on both polymerized and elastase-complexed molecular forms of AAT. The antibody has no apparent affinity for native, latent, or cleaved forms of AAT. The antibody ATZ11 illustrates the structural resemblance between the polymerized form of AAT and its complex with elastase and provides evidence that Z-homozygotes beyond the native form may have at least one more circulating molecular form of AAT, i.e. its polymerized form. In addition, staining of endothelial cells with ATZ11 antibody in both M- and Z-AAT individuals shows that AAT attached to endothelial cells is in a polymerized form. The antibody can be a powerful tool for the study of the molecular profile of AAT, not only in Z-deficiency cases but also in other (patho)physiological conditions.     

Protection against Campylobacter jejuni infection in suckling mice by anti-flagellar antibody

We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.